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† Corresponding author 
© 2015 Conscientia Beam. All Rights Reserved. 

 

EVALUATION OF IN VITRO PROTOCOLS FOR ELIMINATION OF BANANA 

STREAK VIRUS FROM TISSUE CULTURED EXPLANTS IN BANANA 

SEEDLING PRODUCTION  

 

G. Mungai1† --- E. Ateka2 --- A. Nyende3 --- D. Miano4 
1,2,3 Jomo Kenyatta University of Agriculture and Technology (JKUAT) Nairobi Kenya, Kenya 

4Kenya Agricultural Research Institute (KARI), National Agricultural Research Laboratories (NARL), Kenya 

 

ABSTRACT 

The banana industry in Kenya is threatened by the presence of Banana streak virus (BSV). The Jomo 

Kenyatta University of Agriculture and Technology (JKUAT) commercial banana laboratory uses tissue 

culture (TC) technique for mass propagation of plantlets which are free from most disease causing 

organisms for commercial purposes. To evaluate in vitro protocols for production of Banana Streak Virus-

free TC banana planting materials for farmers, leaf samples were collected from Thika, Kisii, and JKUAT 

orchards for indexing. The corms were taken through the TC procedure up to the 2nd subculture stage after 

which they were subjected to three virus elimination techniques; chemotherapy, meristem tip culture and 

thermotherapy for evaluation. Indexing for BSV using PCR BSV indicated 90, 80 and 40% infection levels 

for Kisii, JKUAT and Thika orchards, respectively. For chemotherapy evaluation, concentrations of 

between 10 and 40 mg/l were used resulting in 0 to 90% virus elimination. For thermotherapy, 27°C 

(control), 32°C, 34°C, 36°C and 38°C for 10 days, resulted in 0 and 90% virus elimination. Meristem tip 

culture at 1, 2, 3, 4 and 5mm (control) gave between 0 and 90% virus elimination, respectively. The study 

indicates that BSV can be eliminated using chemotherapy, thermotherapy and meristem tip culture.  

Chemotherapy using salicylic acid at 20mg/l can be used to eliminate BSV up 90%. It is also easy to 

implement since it is incorporated into the medium.      

Keywords: Banana streak virus, Chemotherapy, Thermotherapy, Meristem tip 

 

Contribution/ Originality 

This study contributes to the existing literature on virus elimination from banana planting 

materials globally and the first logical analysis in the fight against banana streak virus in Kenya.  

If adopted, the findings of the study can help banana farmers increase the yield translating to 

higher income and poverty eradication. 

 

Current Research in Agricultural Sciences 
2015 Vol. 2, No. 3, pp. 81-89 
ISSN(e): 2312-6418 
ISSN(p): 2313-3716 
DOI: 10.18488/journal.68/2015.2.3/68.3.81.89 
© 2015 Conscientia Beam. All Rights Reserved. 

 
 
 
 
 

http://crossmark.crossref.org/dialog/?doi=10.18488/journal.68/2015.2.3/68.3.81.89


Current Research in Agricultural Sciences, 2015, 2(3): 81-89 

 

 

82 
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1. INTRODUCTION 

In Kenya, banana is grown for home consumption and for the national market [1]. It is also 

perceived as a major avenue to alleviate food insecurity in the region [2].  Despite this, optimum 

banana yields are lowered by banana streak disease (BSD) caused by Banana streak virus (BSV). 

BSV is prevalent in all banana growing regions in Kenya and all popular cultivars grown by 

farmers are susceptible [3]. The JKUAT banana tissue culture (TC) laboratory is one of the 

biggest in Kenya among others like Mimea limited and KARI Thika, producing over two million 

plantlets per year [4]. The propagation method in use at the JKUAT commercial laboratory is 

micro propagation of banana corms through tissue culture. This method ensures production of 

sufficient planting materials which are free from most disease causing organisms but are not free 

from banana viruses. The JKUAT TC laboratory supplies planting materials all over the country 

and the East African region including Ethiopia, Southern Sudan and Somalia [4].  

Initiation of up to 400, 100, and 100 pieces from JKUAT, Kisii and Thika orchards, 

respectively are used in the TC laboratory every month. It is therefore important to screen for the 

presence of viruses in initiation materials before using for massive TC plantlets production. 

Banana streak virus-infected banana plants frequently express broken or continuous chlorotic or 

necrotic streaks on the leaves, stunting of diseased plants and occasionally heart-rot of the 

pseudostem and plant death. However, the disease symptoms may vary depending on virus strain, 

host genotype, level of management and environmental conditions Harper, et al. [5], Dahal, et al. 

[6], and Lockhart and Jones [7].  Banana streak virus causes yield loss of up to 90% [8], [9].  

There are many BSV elimination methods in use today which include chemotherapy (the use 

chemicals such as Ribavirin and salicylic acid), thermotherapy and meristem tip culture which 

involves the use of the apical dome or shoot tip with a few leaf primordia of the size less than 

2mm in length as explant, cryotherapy and electrotherapy [10]. In this study, three virus 

elimination methods namely chemotherapy, thermotherapy and meristem tip culture were 

evaluated to determine their effectiveness in eliminating BSV from infected banana explants.  

 

2. MATERIALS AND METHODS 

2.1. Sampling Sites  

Three orchards were selected from four different banana orchards, namely Kisii, Thika and 

JKUAT for leaf and sucker samples. Nusu Ng’ombe, Kampala and Fhia 18 varieties were 

randomly sampled (five symptomatic and five asymptomatic) from Kisii, JKUAT and Thika 

orchards respectively.   

 

2.2. Nucleic Acid Extraction, PCR and Gel Electrophoresis  

Total genomic DNA extraction from the leaf samples was done using the CTAB method as 

described by Doyle and Doyle [11].  Nucleic acid quantification was done by observing the bands 

on 0.8% agarose gel (0.8 g agarose gel dissolved in 100ml of Tris boric EDTA (1 X TBE) to 

confirm presence and quality of nucleic acid. The purity and nucleic acid concentration was 

determined by measurement of the absorbance at 260 and 280 nm in a spectrophotometer.  



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Five specific primer pairs (Table 1.0) for detecting each BSV strain were used for detecting 

the presence of the various BSV strains in the banana leaves. PCR mixes (20 μL) containing 10 μl 

2x GoTaq Green Master Mix (Promega Corp, Madison, WI), 5 ρmol of each primer, 1 μl of 

nucleic acid extract and water to final volume. Polymerase chain reaction (PCR) cycling 

conditions included an initial denaturation of 94°C for 2 min followed by 35 cycles at 94°C for 20 

s, 57°C for 20 s, and 72°C for 30 s, with a final extension at 72°C for 2 min. Reaction products 

were analyzed by agarose gel electrophoresis and amplicons visualized in 1.5% agarose gel. 

 
Table-1. Primer pairs used in the amplification of various BSV strains 

Primer name Primer sequence Gene bank Accession 
No1 

Virus strain2 Fragment  length 
(in base pairs) 

RD-F1 5’-ATCTGAAGGTGTGTTGATCAATGC-3’ AF215816 BSV-RD 522 

RD-R1 5’-GCTCACTCCGCATCTTATCAGTC-3’ AF215816 BSV-RD 522 

Cav-F1 5’-AGGATTGGATGTGAAGTTTGAGC-3’ AF215815 BSV-Cav 782 

Cav-R1 -r 5’-ACCAATAATGCAAGGGACGC-3’ AF215815 BSV-Cav 782 

GF-F1 5’ACGAACTATCACGACTTGTTGTTCAAGC-
3’ 

AF215814 BSV-GfV 476 

GF-R1 5’-TCGGTGGAATAGTCCTGAGTCTTC-3’ AF215815 BSV-GfV 476 

BSV4673-F1 5’-GGAATGAAAGAGCAGGCC-3’ AJ002234 BSOEV 644 

BSV5317-R1 5’-GGAATGAAAGAGCAGGCC-3’ 
5’-AGTCATTGGGTCAACCTCTGTC-3’ 

AJ002234 
 

BSVOEV 644 

1A-F1 5’-CTNTAYGARTGGYTNATGCCNTTYGG-
3’ 

AY189378 Badna 597 

4’-R1 5’-TCCAYTTRCANAYNSCNCCCCANCC-3’ AY189383 Badna 597 
 

Adopted from Geering, et al. [12], Harper, et al. [13] BSV-RD = BSV Red Dacca, BSV- Cav= BSV Cavendish, BSV- GfV= BSV Gold 
Finger,  
BSV- BSOEV= BSV – Mysore, Badna = degenerate primer  
1Represents the accession number of the sequence used for primer designing 
 2Represents the name of the virus strain from which primers was designed 

 

2.3. Multiplication of BSV Infected Plantlets   

Suckers were uprooted from the three mother orchards supplying JKUAT banana 

commercial laboratory with planting materials and taken in the laboratory. They were surface 

sterilized  using 70% ethanol for about 8 to 10 seconds after which they were transferred for 20 

minutes in 70% sodium hypochlorite where three drops of tween 20 (wetting agent) had been 

added, the explants were then rinsed twice using double distilled water in the clean bench.  

The scorched outer coverings caused by the sodium hypochlorite were removed using sterile 

surgical blades, leaving a cube of about 2.5 cm3. Using sterile forceps, the explants were placed in 

500ml3 jam jars containing semisolid MS medium. Cultures were incubated at 26 ± 1°C under 

photoperiod cycles of 16 hour under light and 8 hours dark. Light intensity of 25000 lux, from 

white fluorescent tubes was used. The explants were sub cultured after every 4 weeks to fresh MS 

multiplication medium until they reached the 2nd subculture while those explants that were BSV 

infected were subjected to virus elimination methods namely chemotherapy, thermotherapy, and 

meristematic tip culture. The infected banana explants were cultured in Murashige and Skoog  

medium with specification [14] for multiplication.  

 

2.4. Chemotherapy, Thermotherapy and Meristem Tip Culture of BSV-Infected Plantlets 

The plantlets were transferred to fresh MS medium after every 4 weeks to obtain enough TC 

material for the purposes of eliminating the virus. After the second subculture, the plantlets were 



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subjected to the three virus elimination techniques namely chemotherapy, using two chemicals 

(ribavirin and salicylic acid) at 10, 20, 30 and 40 mg/l concentrations, thermotherapy at 32°C, 

34°C, 36°C and 38°C and 27°C as control and meristematic tip culture using 1, 2, 3, 4, and 5mm 

size explants. In chemotherapy, In vitro meristems tips (5mm long) at the 2nd subculture were 

excised in a laminar flow hood and cultured in MS medium supplemented with 0 (control), 10, 20, 

30 and 40 mg /l of ribavirin. Another set of cultures were supplemented with 0 (control), 10, 20, 

30 and 40 mg/l of salicylic acid. After 4 weeks of incubation in the laboratory, the leaves were 

removed using sterile surgical blades in a laminar flow hood, and put in well labelled packing 

bags then stored in a freezer for virus indexing.  In thermotherapy, explants at 2nd stage sub-

culture with well-formed shoots were incubated at varying temperatures, 27°C (control), 32°C, 

34°C, 36°C and 38°C for ten days. The leaves were then removed using sterile surgical blades in a 

laminar flow hood, and placed in well labelled packing bags and then stored in a freezer for PCR 

detection of BSV. In meristem tip culture, at the 2nd sub-culture, explants of 1mm, 2mm, 3mm, 

4mm and 5mm (control) in size were excised using clean surgical blades in the clean bench, before 

being inoculated onto petri dishes containing MS medium. They were left for 2 to 3 days after 

which they were transferred to jars containing 500mls hormone free MS medium to enhance 

shoot elongation, for 4 weeks.  The explants were then transferred to jars containing fresh MS 

medium supplemented with 10mg/l NAA to enhance root formation and the leaves were put in 

well labeled packing bags and stored at –20° C. DNA was extracted using CTAB method 

according to Doyle and Doyle [11] and DNA visualized in 0.8% agarose gel. Once the quantity of 

DNA was confirmed, PCR was done to determine whether the virus had been eliminated. 

 

2.5. Data Collection and Analysis 

The number of explants that survived and number of plantlets without the BSV was recorded 

after every four weeks in spread sheets and before the analysis, the collected data was converted 

into percentages then they were analyzed using the generalized linear model using the Poisson 

log linear model.  

 

3. RESULTS 

3.1. Detection BSV Infection in Banana Plantlets by PCR  

All BSV- specific strains were detected in all the samples except BSV- Cavendish. Double 

infections of BSV strains were observed in three samples while one Sample had three BSV strains 

(Fig 1).  

Only Red Dacca and Gold Finger strains were amplified when PCR was performed on Fhia 

18 samples.  Out of the ten samples amplified, five had at least one BSV strain infection (Table 

2.0).  

In Nusu Ng’ombe variety, PCR analysis using the five BSV specific primers, showed that out 

of the ten samples tested, nine had at least one or more BSV strain detected (Table 2.0). 

 



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Fig-1. Detection of Banana streak virus (BSV) in banana variety Kampala using four specific primers. The four specific 
primers detected the virus in eight out of the ten samples tested, BSV Mysore (589bp), BSV –RD (522bp), BSV Gold 
Finger (476bp).  Lane 1-10: BSV Mysore, lane 11-20: BSV Red Dacca, lane 21-29: BSV Gold Finger, lane M: molecular 
weight marker 

 

No BSV– Cavendish strain was detected in this variety while the most prevalent BSV strain 

from Nusu Ng’ombe variety was Red Dacca (522bp), which was amplified in seven out of ten 

samples tested. BSV- Gold Finger (476bp) strain was amplified in three samples. Three samples 

had double infections while the other six had only one infection. When the degenerate primer 

(Badna-596bp) was used,  BSV strains were detected in all samples except in 56K.  

 

Table-2. Detection of BSV in mother orchards by PCR 

Orchard Variety % BSV infection detection  via PCR 

JKUAT Kampala 80 
KISII Nusu Ng’ombe 90 

THIKA Fhia 18 50 

 

3.2. Virus Elimination  

The BSV infected samples confirmed by PCR were subjected to the three virus elimination 

techniques and the virus eliminations recorded (Table 3, 4 and 5). For chemotherapy, the higher 

the concentration of the chemicals used (Ribavirin and Salicylic acid), the higher the percentage in 

virus elimination in all the three banana varieties (DF=1; P < 0.05). For thermotherapy, there 

was 100% survival in all the temperatures used except at 38°C in which all the explants were 

scorched. Higher virus elimination was recorded at 36°C (DF=2; P<0.01) (Table 4.0). For 

meristem tip culture, the smaller the size of the explants, the higher the percentage in virus 

elimination. However, the smaller the size of the explants the lower was the regeneration of the 

explants (DF= 4; P < 0.001), (Table 5.0)  

 

3.2.1. Chemotherapy  

In chemotherapy, variety, chemical and chemical concentration had an effect on survival of 

the explants and virus elimination. (Table 3.0, 4.0, 5.0) 

 

3.2.1.1. Effect of Variety on Survival of Explants and BSV Elimination  

Variety used had an effect on virus elimination and survival (df=; P<0.05).  Nusu Ng’ombe 

variety had the highest 80.9±2.35 BSV elimination against Kampala and Fhia 18 at 73.3±5.42 and 

80.8±2.16 respectively using the same chemicals and same chemical concentrations. Variety Nusu 



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Ng’ombe had the highest mean of the surviving explants of 65.65 against Kampala and Fhia 18 at 

62.6 ±6.01 and 57.92 ±7.57 respectively (Df=2; P<0.05) (Table 3.0) 

 

Table-3. Effect of variety on survival of explants and BSV elimination 

Variety  Survival of explants Virus elimination  

Fhia 18 57.92+7.566 80.83+2.163 

Kampala 62.59+6.006 73.33+5.417 

Nusu Ng’ombe  65.65+6.090 80.87+2.345 

         There was an effect of variety used on survival and BSV elimination (DF=2, P<0.05).  

 

3.2.1.2. Effect of Chemical on Survival of Explants and Virus Elimination  

Even though both chemicals eliminated the virus, elimination using salicylic acid was higher 

than that of ribavirin (DF=2; P<0.05) Salicylic acid had a higher survival mean of 62.1 ± 3.97 

compared to ribavirin at 61.9 ± 5.18 salicylic acid had a higher BSV elimination of 81.1 ± 1.816b% 

as compared to 75.26 ± 3.969a% across the three varieties and chemical concentrations (Table 

4.0). 

 

Table 4. Effect of chemical on survival of explants and virus elimination 

Chemical  survival BSV elimination 

Ribavirin  61.94 ± 5.177 75.26 ± 3.969 

Salicylic acid 62.11 ± 3.969 81.11 ± 1.816 

 

3.2.1.3. Effect of Chemical Concentration to Survival of Explants and BSV elimination  

At 0mg/l, survival of explants was as higher as 96.8+3.33 with 0.0+0.0 BSV elimination. 

When concentration was increased, survival of explants reduced with increase in BSV elimination. 

At 10, 20, 30 and 40mg/l had 83, 78, 66 and 11explants survival and 76, 82, 84 and 88 BSV 

elimination respectively (Table 5.0) (DF=4; P<0.05) 

 

Table 5. Effect of chemical concentration on survival of explants and BSV elimination 

Chemical concentration (mg/l) Explant Survival  BSV Elimination 

0 96.67+3.333 0.00+0.00 

10 83.33±3.052 76.67±2.567 

20 78.33±3.052 82.78±1.948 

30 66.11±5.248 84.44±2.318 

40 11.76±2.461 88.76±0832 

     Chemical concentration had a significant effect on explant survival and BSV elimination at DF= 4; P<0.05) 

 

3.2.2. Thermotherapy  

3.2.2.1. Effect of Variety on BSV Elimination and Survival of Explants  

The variety had a significant effect on both BSV elimination from the explants (df=2; 

P<0.001) Kampala variety had the highest BSV elimination (39.6 ±10.86 while Nusu Ng’ombe 

had the lowest virus elimination (37.9±10.93) at the same temperatures (Table 6.0). 



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Table 6. Effect of variety to BSV elimination and survival of explants 

Variety Virus elimination Survival 

Fhia 18 39.17±10.88a 100.00±0.00a 

Kampala 39.58±10.86a 100.00±0.00a 

Nusu Ng’ombe 37.92±10.93a 100.00±0.00a 

            Variety used had a significant effect on both BSV elimination from the explants (df=2; P<0.001) 

 

3.2.2.2. Effect of Temperature on BSV Elimination and Survival of Explants  

Temperature had a significant effect on BSV elimination as well as survival of explants 

(p<0.05). The higher temperature, contributed to higher BSV elimination. All temperature levels 

used had a 100.0 ± 0.00 explants survival except when the explants were placed at 38°C when all 

explants got scorched The lower the temperature, the higher the BSV elimination ( Table 7.0).  

 

Table 7. Effect of Temperature on BSV elimination and survival of explants 

Temperature (°C) Virus elimination Survival 

27 0.00±0.00 100.00±0.00 

32 13.33± 6.47 100.00±0.00 

34 57.78±5.54 100.00±0.00 

36 84.89±1.54 100.00±0.00 

38 n/a died 

          Temperature had a significant effect on BSV elimination (Df =4; P<0.05) 

 

3.2.3. Meristem Tip Culture  

3.2.3.2. Effect of Meristem Tip Size on BSV Elimination and Explants Survival  

The highest virus elimination was at 1mm sized explants. The smaller the explant used the 

lower the survival and the higher the BSV elimination and vice versa in all the three varieties. At 

1mm, BSV elimination was 89.0±1.20 and 32.4±0.38 plants survival respectively, (DF = 4; 

P<0.001) (Table 9.0) 

 

Table 9. Effect of meristem tip size on BSV elimination and explants survival 

Size (mm) BSV elimination  Explants survival 

1 89.00±1.202a 32.44±0.377b 

2 62.56±0.766a 100.00±0.00a 

3 43.00±3.00b 100.00±0.00a 

4 0.56±0.242c 100.00±0.00a 

5 0.11±0.11c 100.00±0.00a 

The size of the explant used had a significant effect on both the BSV elimination and survival of the explants in all the varieties (Df=4; 

P<0.001). 

 

4. DISCUSSION  

All the three methods evaluated for elimination of Banana streak virus were effective though 

at different specifications. Heat therapy gave the best results at 36°C, while chemotherapy gave 

the best results at 20mg/l for both chemicals used separately and meristem tip culture at 1 mm .  



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The different specific primers pairs (Table 1.0) used to detect different strains of BSV 

amplified all the BSV strains present except for BSV Cavendish. Results of this work showed that 

50% of the samples collected were BSV infected (Table 2.0). These results are in agreement with 

previous studies carried out in Kenya which showed that BSV is prevalent in all banana growing 

regions and that all popular cultivars grown by farmers are susceptible [15], [16], [3]. 

 In meristem tip culture, the size of the explant used had a significant effect on both explant 

survival and BSV elimination. The smaller the size of the meristem used the higher the BSV 

elimination (Table 8.0 and 9.0).  

The success of heat therapy depends on selecting the temperature and the duration of the 

treatment for the elimination of the virus and also with the survival of the plant. In this study, all 

explants exposed to temperatures of  27°C, 32°C, 34°C and 36°C survived after being put in an 

incubator for 10 days, but at 38°C all the explants were scorched by the heat and therefore no 

plantlet could be regenerated from this treatment. The highest virus elimination percent was at 

36°C ( Table 6.0 and Table 7.0).  

The results demonstrate that ribavirin and salicylic acid at concentration of 10mg/l enhanced 

growth differentiation of propagated meristems.  It can be concluded that the use of ribavirin and 

salicylic acid  separately at 20 mg/l combined with TC process can increase the percentage of 

virus-free plants.  It was noted that the higher the  chemical concentration , the higher the virus 

elimination percentage (Table 5.0). This results are in agreement with those obtained by 

Qiaochunand, et al. [10].  

Although, thermal treatment at 36°C resulted in high viral elimination and survival rate of 

explants treated, the technique can be cumbersome for commercial laboratories leaving 20mg/l of 

salicylic acid as the best option. This is because it is easy to incorporate the chemical into the 

medium  used  in TC and it is far much cheaper than thermotherapy which requires an incubator 

and frequent monitoring. 

When TC procedures without any of the three virus elimination techniques was used as 

control, there were no viral elimination which is in agreement [15], [3] that TC technique does 

not eliminate Banana streak virus. Therefore, though TC has very many advantages over the 

conventional methods of mass propagation, it does not eliminate BSV. Therefore, a combination 

of TC with a reliable virus elimination method is required to be put  in place to be able to 

eliminate BSV. 

 

5. CONCLUSIONS AND RECOMMENDATIONS    

This study demonstrated that Banana streak virus can be eliminated using chemotherapy, 

thermotherapy and meristem tip culture. Chemotherapy using salicylic acid at 20mg/l was 

selected as the best method with up to 90% BSV elimination. The method is also easy to 

implement since it is incorporated into the medium. It is recommended that banana streak virus 

free orchards be established and continuous checks be done to ensure only BSV plantlets are in 

circulation. 

 



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